The cardiac voltage-gated sodium channel Na1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium ... More
The cardiac voltage-gated sodium channel Na1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Na1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na1.5 at the cell surface and in whole-cell lysates. Localization of Na1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na1.5 NTD using modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na1.5 variant Y87C and present first evidence that calmodulin binds to the Na1.5 NTD, which seems to be a determinant for the DNE.
